The theme of this Program Project proposal is to develop novel therapies for hemophilia. In particular, the focus will be on the most significant complication of the disease, namely the occurrence of inhibitory antibodies to the clotting factors that are normally used to treat bleeding episodes. The development of an inhibitory is a devastating event, which leads inevitably to increased morbidity, and cost of therapy (which is already exceptionally high for patients without this complication). Furthermore, development of inhibitors remains one of the principal concerns surrounding gene therapy for hemophilia. We will adopt a multi- disciplinary approach to this problem, concentrating on bio-engineered clotting factors (mutants of factor VIIa with enhanced pro-coagulant activity, and human-porcine factor VIII hybrid molecules with reduced immunogenicity and antigenicity. Although not part of this proposal, clinical trials involving these proteins can be realistically anticipated as a longer term goal. In Project 1 ("The Role of Tissue trials involving these proteins can be realistically anticipated as a longer term goal. In Project 1 ("The Role of Tissue Factor in Hemophilia"), we will examine the biochemical basis for tissue factor encryption in vitro, and develop methods to measure expression of both the encrypted and pro-coagulant forms of tissue factor in vivo. In Project ("Enhanced Vitamin K- dependent Proteins in Hemophilia"), recombinant factor VII molecules that have been mutated at specific residues in the membrane contact region will be characterized with respect to their pro-coagulant activity. The anti-hemorrhagic efficacy (and potential undesirable thrombogenicity) of selected mutants will be tested in animal models of hemophilia. In Project 3 ("CD4+ T Cell Response to Porcine Factor VIII"), CD4+ T cell responses to human-porcine hybrid FVIII molecules will be examined, primarily in the murine model of hemophilia A with a FVIII inhibitor. It is our hypotheses that these hybrid molecules may be less immunogenic than human FVIII. Finally, in Project 4 ("Chimeraplasty for Factor IX and Factor VII Gene Expression"), we will use chimeric RNA/DNA constructs to "repair" the point mutation in the factor IX gene in canine hemophilia, and deliberately induce a selected point mutation in the factor VII gene to promote expression of high activity FVII(a) in vivo that will "by-pass" the need for factor VIII (or IX) in patients with inhibitors.